Elucidating the Mechanisms of Lipid Droplet Protein Degradation

项目总结 日益严重的代谢性疾病全球流行是一个紧迫的公共卫生问题。就像流行的 肥胖、胰岛素抵抗和非酒精性脂肪肝等疾病继续攀升，需要 深入了解细胞内脂肪的储存机制已变得越来越迫切。多数 代谢紊乱涉及脂肪在肝脏和心脏等组织中的异常积聚，导致 对全身健康造成毁灭性的影响。在细胞内，脂类储存在称为脂滴的细胞器中。 (LDS)，由三酰甘油和以磷脂为边界的胆固醇酯组成的中性脂核 单层。与单层相关的是控制动态的多种调节蛋白和酶 封存和释放脂类储备，使LD蛋白影响整个 手机。尽管LD蛋白在维持细胞内脂平衡方面起着重要作用，但关于 LD蛋白本身的调节--特别是控制LD蛋白丰度的途径。 尽管有几项研究报道了泛素-蛋白酶体系统(UPS)在调节LD蛋白水平中的作用， 所需泛素化机制的同一性(例如E3泛素连接酶和E2泛素结合 酶)以及它们发挥控制作用的途径仍不清楚。要解决这些基本问题 问题，我建议1)一个全基因组的、基于荧光的CRISPR/Cas9筛查来识别降解 LD蛋白Perilipin-2(PLIN2)的途径，以及2)询问PLIN2如何受损的随访研究 退化影响全球细胞新陈代谢。我的特征是一种人类肝癌Cas9-表达 GFP标记内源性PLIN2的荧光报告细胞系。流式细胞术，Western blotting， 荧光显微镜分析证实PLIN2-GFP在内源性水平表达，定位于 到LDS，并被蛋白酶体降解。验证的PLIN2-GFP细胞系被用于中试筛选 一个每基因10个引导的慢病毒单导引RNA亚库(SgRNAs)，富含UPS基因。此屏幕 确定了几个候选UPS因素，我假设这些因素与PLIN2的降解有关。我的建议 研究包括完成全面的全基因组筛查，验证候选基因， PLIN2降解途径的特征，以及受损的功能后果的检查 Plin2间隙。这些研究将阐明UPS调节LD蛋白的机制，提供 对细胞如何维持脂质动态平衡的新见解。了解LD蛋白质降解途径将 能够理解LD蛋白调节的改变如何有助于代谢的发病机制 疾病。

PROJECT SUMMARY The growing global epidemic of metabolic disease is a pressing public health issue. As the prevalence of disorders such as obesity, insulin resistance, and nonalcoholic fatty liver continue to climb, the need for a thorough understanding of cellular lipid storage mechanisms has become increasingly imperative. Most metabolic disorders involve the aberrant accumulation of lipid in tissues such as the liver and heart, leading to devastating systemic health effects. Within cells, lipids are stored in cytosolic organelles called lipid droplets (LDs), which consist of a neutral lipid core of triacylglycerols and cholesterol esters bounded by a phospholipid monolayer. Associated with the monolayer are multiple regulatory proteins and enzymes that control the dynamic sequestration and release of the lipid reserves, allowing LD proteins to influence the metabolism of the entire cell. Although LD proteins have essential roles in maintaining cellular lipid homeostasis, little is known regarding the regulation of LD proteins themselves – in particular, the pathways that control LD protein abundance. Although several studies report a role for the ubiquitin-proteasome system (UPS) in modulating LD protein levels, the identities of the required ubiquitination machinery (e.g. E3 ubiquitin ligases and E2 ubiquitin conjugating enzymes) and the pathways by which they exert control remain unclear. To address these fundamental questions, I propose 1) a genome-wide, fluorescence-based CRISPR/Cas9 screen to identify the degradation pathway of the LD protein perilipin-2 (PLIN2), and 2) follow-up studies to interrogate how impaired PLIN2 degradation impacts global cellular metabolism. I have characterized a human hepatoma Cas9-expressing fluorescent reporter cell line in which endogenous PLIN2 is tagged with GFP. Flow cytometry, Western blotting, and fluorescence microscopy analyses confirmed that PLIN2-GFP is expressed at endogenous levels, localizes to LDs, and is degraded by the proteasome. The validated PLIN2-GFP cell line was employed in a pilot screen of a 10-guide-per-gene lentiviral sublibrary of single guide RNAs (sgRNAs) enriched in UPS genes. This screen identified several candidate UPS factors that I hypothesize are involved in PLIN2 degradation. My proposed studies include the completion of a comprehensive, genome-wide screen, validation of candidate genes, characterization of PLIN2 degradation pathways, and examination of the functional consequences of impaired PLIN2 clearance. These studies will elucidate the mechanisms of LD protein regulation by the UPS, providing novel insights into how cells maintain lipid homeostasis. Knowledge of LD protein degradation pathways will allow for an understanding of how alterations in LD protein regulation contribute to the pathogenesis of metabolic disease.